Human gammaherpesvirus infection is linked to the development of multiple types of malignancies. These viruses pose a particularly significant risk to immunocompromised patients such as those infected with HIV. Gammaherpesviruses establish stable life-long latent infection in B cells, providing a lifelong reservoir of virus that can eventually contribute to the development of malignant disease. Our long-term goal is to define the mechanisms that gammaherpesviruses use to establish latency in B cells, thereby elucidating new targets for the development of modalities to prevent gammaherpesvirus-associated malignancies. Our approach is to dissect the earliest events of latency establishment using mutant viruses in the murine gammaherpesvirus (gammaHV68, MHV68) system that are unable to undergo lytic replication. By comparing viruses that are mutated in early versus late lytic genes, we have uncovered a surprising role for lytic replication-associated gene products in the establishment of latency in B cells. We hypothesize that these key lytic gene products are critical for the induction of epigenetic modifications that are critical for retention of latent viral episomes. The goals of this proposal are to define the critical molecular events that occur in the earliest hours following B cell infection that ultimately result in stable maintenance of the viral episome during latency. Specifically, we will define the viral transcriptional and genomic events that occur during the establishment phase of B cell latency (Aim 1), we will define the specific late lytic gene that is required for the establishment of stable latency (Aim 2), and we will test the hypothesis that the crucial lytic gene induces epigenetic modifications that are critical for latency establishment in B cells. The proposed studies should provide significant new insight into the critical molecular events that occur during the earliest moments of gammaherpesvirus infection of B cells.